Project Summary/Abstract The brain endocannabinoid (eCB) system can dampen behavioral and physiological responses to stress via activation of cannabinoid receptor 1 (CB1), one of the most abundant G protein-coupled receptors in the brain. However, the distinct neural circuits which underlie eCB-mediated effects remain incompletely identified and understood. Recent studies have identified the habenula, an epithalamic structure conserved across vertebrate evolution, to play a central role in encoding stress reactivity, avoidance behavior, and aversion in rodents and non-human primates, and its hyperactivity has been linked to anxiety-like behavior and mood dysregulation. Human studies have shown that habenula volumes and cell numbers are reduced in patients with depressive disorders, and deep brain stimulation of the habenula led to remission in an individual with treatment-resistant depression. Thus, both human and animal studies point to a critical role for the habenula in regulating behaviors relevant to stress responsivity and mood. In this study, we provide first evidence that neurons of the medial habenula (MHb) synthesize and release eCBs to suppress synaptic input from the medial septum and nucleus of the diagonal band (MSDB). Further, we show that this eCB/CB1 signaling is blunted in association with stress-induced anxiety-like behavior, and that knockdown of CB1 receptors in the MSDB is sufficient to mimic the stress-induced behavioral phenotype. Thus, these results, together with previous literature linking the MHb and the MSDB to anxiety-like behavior, led us to hypothesize that eCB/CB1 signaling in the MSDB- MHb pathway is a novel mechanism whereby eCBs attenuate stress-induced anxiety-like behavior, and that dysregulation of this signaling can contribute to this behavioral state. Three Specific Aims are proposed to test these hypotheses. Our first goal in Aim I will be to carry out a detailed assessment of the cell type-specific connectivity of the MSDB-MHb pathway, as very little is known regarding its anatomy and physiology. In Aim II, we will determine how eCBs regulate this circuit and how this regulation is affected by stress exposure. In Aim III, we will test the hypothesis that eCB signaling in this pathway regulates behavioral responses to stress. Successful completion of the outlined studies will advance our long-term objectives to understand the neural circuits underlying anxiety-like behavior, the specific mechanisms whereby eCBs exert behavior effects, and how dysregulation of eCB signaling may contribute to anxiety and mood-related disorders.